The signal recognition particle (SRP) is a highly evolutionarily conserved ribonucleoprotein complex that functions to target proteins into biological membranes. Because E. coli contains a "minimal" SRP, consisting of the Ffh protein in association with a 4.5S RNA species, it provides an attractive system to understand how the translocation machinery functions in all living cells. Understanding the role of the RNA component of the bacterial SRP function is complicated by its essentiality for cell growth and its involvement in protein synthesis. Since few studies have been performed to understand how the sequence determinants of 4.5S RNA contribute to its function in vivo, little is known about why the RNA is an essential component of both the membrane protein localization and protein translation machinery. The long-term goal of our research is to determine how 4.5S RNA functions as a component of the SRP to target proteins to the inner membrane, and in translation by its interaction with elongation factor G. We propose to perform a systematic genetic characterization of 4.5S RNA to determine how the structure of this molecule contributes to its activity, and to probe the function of the RNA in both protein synthesis and membrane protein localization. Our lack of knowledge about the cellular roles of 4.5S RNA is significant in that it prevents us from fully describing the essential processes of protein localization and polypeptide synthesis on a molecular level. Due to the high evolutionary conservation of the SRP RNA, with homologues found in all living species examined to date, we will also better comprehend how this RNA species functions in all living systems. Although recent efforts have been placed largely on in vitro analysis of 4.5S RNA, including biochemical characterization and structural determinations, efforts to understand the function of 4.5 S RNA in vivo has lagged in part due to the lack of genetic systems for studying this molecule. These studies should contribute to improved methods to express and localize membrane proteins of medical importance, as well as lead to the identification of antimicrobial agents.